RESUMO
Integration of DNA viruses into the human genome plays an important role in various types of tumors, including hepatitis B virus (HBV)-related hepatocellular carcinoma. However, the molecular details and clinical impact of HBV integration on either human or HBV epigenomes are unknown. Here, we show that methylation of the integrated HBV DNA is related to the methylation status of the flanking human genome. We developed a next-generation sequencing-based method for structural methylation analysis of integrated viral genomes (denoted G-NaVI). This method is a novel approach that enables enrichment of viral fragments for sequencing using unique baits based on the sequence of the HBV genome. We detected integrated HBV sequences in the genome of the PLC/PRF/5 cell line and found variable levels of methylation within the integrated HBV genomes. Allele-specific methylation analysis revealed that the HBV genome often became significantly methylated when integrated into highly methylated host sites. After integration into unmethylated human genome regions such as promoters, however, the HBV DNA remains unmethylated and may eventually play an important role in tumorigenesis. The observed dynamic changes in DNA methylation of the host and viral genomes may functionally affect the biological behavior of HBV. These findings may impact public health given that millions of people worldwide are carriers of HBV. We also believe our assay will be a powerful tool to increase our understanding of the various types of DNA virus-associated tumorigenesis.
Assuntos
Metilação de DNA , DNA Viral/genética , Genoma Humano , Vírus da Hepatite B/genética , Integração Viral , Alelos , Elementos Alu , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Ilhas de CpG , Epigênese Genética , Loci Gênicos , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Neoplasias Hepáticas/genéticaRESUMO
Diacylglycerol kinases (DGKs) are important regulators of cell signaling and have been implicated in human malignancies. Whether epigenetic alterations are involved in the dysregulation of DGKs in cancer is unknown, however. We therefore analyzed methylation of the promoter CpG islands of DGK genes in colorectal cancer (CRC) cell lines. We found that DGKG, which encodes DGKγ, was hypermethylated in all CRC cell lines tested (n = 9), but was not methylated in normal colonic tissue. Correspondingly, DGKG expression was suppressed in CRC cell lines but not in normal colonic tissue, and was restored in CRC cells by treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC). DGKG methylation was frequently observed in primary CRCs (73/141, 51.8%) and was positively associated with KRAS and BRAF mutations and with the CpG island methylator phenotype (CIMP). DGKG methylation was also frequently detected in colorectal adenomas (89 of 177, 50.3%), which suggests it is an early event during colorectal tumorigenesis. Ectopic expression of wild-type DGKγ did not suppress CRC cell proliferation, but did suppress cell migration and invasion. Notably, both constitutively active and kinase-dead DGKγ mutants exerted inhibitory effects on CRC cell proliferation, migration and invasion, and the wild-type and mutant forms of DGKγ all suppressed Rac1 activity in CRC cells. These data suggest DGKG may play a tumor suppressor role in CRC.
Assuntos
Adenoma/genética , Neoplasias Colorretais/genética , Metilação de DNA , Diacilglicerol Quinase/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Adenoma/patologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/patologia , Diacilglicerol Quinase/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Estadiamento de Neoplasias , Prognóstico , Regiões Promotoras Genéticas , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
BACKGROUND & AIMS: Subgroups of colorectal carcinomas (CRCs) characterized by DNA methylation anomalies are termed CpG island methylator phenotype (CIMP)1, CIMP2, or CIMP-negative. The pathogenesis of CIMP1 colorectal carcinomas, and their effects on patients' prognoses and responses to treatment, differ from those of other CRCs. We sought to identify genetic somatic alterations associated with CIMP1 CRCs. METHODS: We examined genomic DNA samples from 100 primary CRCs, 10 adenomas, and adjacent normal-appearing mucosae from patients undergoing surgery or colonoscopy at 3 tertiary medical centers. We performed exome sequencing of 16 colorectal tumors and their adjacent normal tissues. Extensive comparison with known somatic alterations in CRCs allowed segregation of CIMP1-exclusive alterations. The prevalence of mutations in selected genes was determined from an independent cohort. RESULTS: We found that genes that regulate chromatin were mutated in CIMP1 CRCs; the highest rates of mutation were observed in CHD7 and CHD8, which encode members of the chromodomain helicase/adenosine triphosphate-dependent chromatin remodeling family. Somatic mutations in these 2 genes were detected in 5 of 9 CIMP1 CRCs. A prevalence screen showed that nonsilencing mutations in CHD7 and CHD8 occurred significantly more frequently in CIMP1 tumors (18 of 42 [43%]) than in CIMP2 (3 of 34 [9%]; P < .01) or CIMP-negative tumors (2 of 34 [6%]; P < .001). CIMP1 markers had increased binding by CHD7, compared with all genes. Genes altered in patients with CHARGE syndrome (congenital malformations involving the central nervous system, eye, ear, nose, and mediastinal organs) who had CHD7 mutations were also altered in CRCs with mutations in CHD7. CONCLUSIONS: Aberrations in chromatin remodeling could contribute to the development of CIMP1 CRCs. A better understanding of the biological determinants of CRCs can be achieved when these tumors are categorized according to their epigenetic status.
Assuntos
Cromatina , Neoplasias Colorretais/genética , Ilhas de CpG , DNA Helicases/genética , Metilação de DNA , Proteínas de Ligação a DNA/genética , Mutação , Fatores de Transcrição/genética , Adenoma/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Exoma , Feminino , Inativação Gênica , Marcadores Genéticos , Humanos , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Fenótipo , Análise de Sequência de DNARESUMO
Colorectal cancers (CRCs) exhibit multiple genetic alterations, including allelic imbalances (copy number alterations, CNAs) at various chromosomal loci. In addition to genetic aberrations, DNA methylation also plays important roles in the development of CRC. To better understand the clinical relevance of these genetic and epigenetic abnormalities in CRC, we performed an integrative analysis of copy number changes on a genome-wide scale and assessed mutations of TP53, KRAS, BRAF, and PIK3CA and DNA methylation of six marker genes in single glands isolated from 39 primary tumors. Array-based comparative genomic hybridization (array-CGH) analysis revealed that genomic losses commonly occurred at 3q26.1, 4q13.2, 6q21.32, 7q34, 8p12-23.3, 15qcen and 18, while gains were commonly found at 1q21.3-23.1, 7p22.3-q34, 13q12.11-14.11, and 20. The total numbers and lengths of the CNAs were significantly associated with the aberrant DNA methylation and Dukes' stages. Moreover, hierarchical clustering analysis of the array-CGH data suggested that tumors could be categorized into four subgroups. Tumors with frequent DNA methylation were most strongly enriched in subgroups with infrequent CNAs. Importantly, Dukes' D tumors were enriched in the subgroup showing the greatest genomic losses, whereas Dukes' C tumors were enriched in the subgroup with the greatest genomic gains. Our data suggest an inverse relationship between chromosomal instability and aberrant methylation and a positive association between genomic losses and distant metastasis and between genomic gains and lymph node metastasis in CRC. Therefore, DNA copy number profiles may be predictive of the metastatic behavior of CRCs.
Assuntos
Neoplasias Colorretais/genética , Hibridização Genômica Comparativa/métodos , Idoso , Idoso de 80 Anos ou mais , Aberrações Cromossômicas , Classe I de Fosfatidilinositol 3-Quinases , Análise por Conglomerados , Neoplasias Colorretais/classificação , Neoplasias Colorretais/patologia , Variações do Número de Cópias de DNA , Metilação de DNA , Feminino , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Metástase Neoplásica , Estadiamento de Neoplasias , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras) , Proteína Supressora de Tumor p53/genética , Proteínas ras/genéticaRESUMO
The mitotic checkpoint gene CHFR (checkpoint with forkhead-associated (FHA) and RING finger domains) is silenced by promoter hypermethylation or mutated in various human cancers, suggesting that CHFR is an important tumor suppressor. Recent studies have reported that CHFR functions as an E3 ubiquitin ligase, resulting in the degradation of target proteins. To better understand how CHFR suppresses cell cycle progression and tumorigenesis, we sought to identify CHFR-interacting proteins using affinity purification combined with mass spectrometry. Here we show poly(ADP-ribose) polymerase 1 (PARP-1) to be a novel CHFR-interacting protein. In CHFR-expressing cells, mitotic stress induced the autoPARylation of PARP-1, resulting in an enhanced interaction between CHFR and PARP-1 and an increase in the polyubiquitination/degradation of PARP-1. The decrease in PARP-1 protein levels promoted cell cycle arrest at prophase, supporting that the cells expressing CHFR were resistant to microtubule inhibitors. In contrast, in CHFR-silenced cells, polyubiquitination was not induced in response to mitotic stress. Thus, PARP-1 protein levels did not decrease, and cells progressed into mitosis under mitotic stress, suggesting that CHFR-silenced cancer cells were sensitized to microtubule inhibitors. Furthermore, we found that cells from Chfr knockout mice and CHFR-silenced primary gastric cancer tissues expressed higher levels of PARP-1 protein, strongly supporting our data that the interaction between CHFR and PARP-1 plays an important role in cell cycle regulation and cancer therapeutic strategies. On the basis of our studies, we demonstrate a significant advantage for use of combinational chemotherapy with PARP inhibitors for cancer cells resistant to microtubule inhibitors.
Assuntos
Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Neoplasias/patologia , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Animais , Neoplasias da Mama , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Pontos de Checagem do Ciclo Celular/fisiologia , Desenho de Fármacos , Feminino , Genes Supressores de Tumor/fisiologia , Células HEK293 , Células HeLa , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microtúbulos/efeitos dos fármacos , Microtúbulos/fisiologia , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias de Células Escamosas/tratamento farmacológico , Neoplasias de Células Escamosas/metabolismo , Neoplasias de Células Escamosas/patologia , Poli(ADP-Ribose) Polimerase-1 , Proteínas de Ligação a Poli-ADP-Ribose , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologiaRESUMO
The concept of the CpG island methylator phenotype (CIMP) in colorectal cancer (CRC) is widely accepted, although the timing of its occurrence and its interaction with other genetic defects are not fully understood. Our aim in this study was to unravel the molecular development of CIMP cancers by dissecting their genetic and epigenetic signatures in precancerous and malignant colorectal lesions. We characterized the methylation profile and BRAF/KRAS mutation status in 368 colorectal tissue samples, including precancerous and malignant lesions. In addition, genome-wide copy number aberrations, methylation profiles, and mutations of BRAF, KRAS, TP53, and PIK3CA pathway genes were examined in 84 colorectal lesions. Genome-wide methylation analysis of CpG islands and selected marker genes revealed that CRC precursor lesions are in three methylation subgroups: CIMP-high, CIMP-low, and CIMP-negative. Interestingly, a subset of CIMP-positive malignant lesions exhibited frequent copy number gains on chromosomes 7 and 19 and genetic defects in the AKT/PIK3CA pathway genes. Analysis of mixed lesions containing both precancerous and malignant components revealed that most aberrant methylation is acquired at the precursor stage, whereas copy number aberrations are acquired during the progression from precursor to malignant lesion. Our integrative genomic and epigenetic analysis suggests early onset of CIMP during CRC development and indicates a previously unknown CRC development pathway in which epigenetic instability associates with genomic alterations.
Assuntos
Neoplasias Colorretais/genética , Ilhas de CpG/genética , Metilação de DNA/genética , Lesões Pré-Cancerosas/genética , Idoso , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Neoplasias Colorretais/patologia , Variações do Número de Cópias de DNA/genética , Progressão da Doença , Endoscopia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genoma Humano/genética , Humanos , Masculino , Mutação/genética , Fenótipo , Lesões Pré-Cancerosas/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras) , Proteínas ras/genéticaRESUMO
DNA methyltransferase 1 (Dnmt1) is essential for the maintenance of hematopoietic and somatic stem cells in mice; however, its roles in human cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are still elusive. In the present study, we investigated DNMT1 functions in the maintenance of human colon CSCs/CICs using the human colon cancer cell line HCT116 (HCT116 w/t) and its DNMT1 knockout cell line (DNMT1(-/-)). The rates of CSCs/CICs were evaluated by side population (SP) analysis, ALDEFLUOR assay and expression of CD44 and CD24. SP, ALDEFLUOR-positive (ALDEFLUOR(+)) and CD44-positive and CD24-positive (CD44(+)CD24(+)) cell rates were lower in DNMT1(-/-) cells than in HCT116 w/t cells. Since CSCs/CICs have higher tumor-initiating ability than that of non-CSCs/CICs, the tumor-initiating abilities were addressed by injecting immune deficient (NOD/SCID) mice. DNMT1(-/-) cells showed less tumor-initiating ability than did HCT116 w/t cells, whereas the growing rate of DNMT1(-/-) cells showed no significant difference from that of HCT116 cells both in vitro and in vivo. Similar results were obtained for cells in which DNMT1 had been transiently knocked-down using gene-specific siRNAs. Taken together, these results indicate that DNMT1 is essential for maintenance of colon CSCs/CICs and that short-term suppression of DNMT1 might be sufficient to disrupt CSCs/CICs.
Assuntos
Transformação Celular Neoplásica , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , DNA (Citosina-5-)-Metiltransferases/metabolismo , Células-Tronco Neoplásicas/fisiologia , Animais , Antígeno CD24/biossíntese , Neoplasias do Colo/genética , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Receptores de Hialuronatos/biossíntese , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Interferência de RNA , RNA Interferente Pequeno , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Aberrant WNT pathway signaling is an early progression event in 90% of colorectal cancers. It occurs through mutations mainly of APC and less often of CTNNB1 (encoding beta-catenin) or AXIN2 (encoding axin-2, also known as conductin). These mutations allow ligand-independent WNT signaling that culminates in abnormal accumulation of free beta-catenin in the nucleus. We previously identified frequent promoter hypermethylation and gene silencing of the genes encoding secreted frizzled-related proteins (SFRPs) in colorectal cancer. SFRPs possess a domain similar to one in the WNT-receptor frizzled proteins and can inhibit WNT receptor binding to downregulate pathway signaling during development. Here we show that restoration of SFRP function in colorectal cancer cells attenuates WNT signaling even in the presence of downstream mutations. We also show that the epigenetic loss of SFRP function occurs early in colorectal cancer progression and may thus provide constitutive WNT signaling that is required to complement downstream mutations in the evolution of colorectal cancer.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Inativação Gênica , Glicoproteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas WntRESUMO
BACKGROUND AND AIMS: The majority of gastrointestinal stromal tumors (GISTs) have KIT mutations; however, epigenetic abnormalities that could conceivably potentiate the aggressiveness of GISTs are largely unidentified. Our aim was to establish epigenetic profiles associated with the malignant transformation of GISTs. METHODS: Methylation of four tumor suppressor genes, RASSF1A, p16, CDH1, and MGMT was analyzed in GISTs. Additionally, genome-wide DNA methylation profiles were compared between small, malignant-prone, and malignant GISTs using methylated GpG island amplification microarrays (MCAM) in a training set (n=40). Relationships between the methylation status of genes identified by MCAM and clinical features of the disease were tested in a validation set (n=75). RESULTS: Methylation of RASSF1A progressively increased from small to malignant GISTs. p16 was specifically methylated in malignant-prone and malignant GISTs. MCAM analysis showed that more genes were methylated in advanced than in small GISTs (average of 473 genes vs 360 genes, respectively, P=0.012). Interestingly, the methylation profile of malignant GISTs was prominently affected by their location. Two genes, REC8 and PAX3, which were newly-identified via MCAM analysis, were differentially methylated in small and malignant GISTs in the training and validation sets. Patients with methylation of at least REC8, PAX3, or p16 had a significantly poorer prognosis (P=0.034). CONCLUSION: Our results suggest that GIST is not, in epigenetic terms, a uniform disease and that DNA methylation in a set of genes is associated with aggressive clinical behavior and unfavorable prognosis. The genes identified may potentially serve as biomarkers for predicting aggressive GISTs with poor survivability.
Assuntos
Metilação de DNA , DNA de Neoplasias/genética , Neoplasias Gastrointestinais/genética , Tumores do Estroma Gastrointestinal/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Proteínas de Ciclo Celular/genética , Ilhas de CpG/genética , Progressão da Doença , Epigênese Genética , Feminino , Neoplasias Gastrointestinais/diagnóstico , Neoplasias Gastrointestinais/patologia , Tumores do Estroma Gastrointestinal/diagnóstico , Tumores do Estroma Gastrointestinal/patologia , Genes Supressores de Tumor , Genes p16 , Estudo de Associação Genômica Ampla , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/genética , Prognóstico , Proteínas Proto-Oncogênicas c-kit/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
OBJECTIVES: Sessile serrated adenomas (SSAs) are known to be precursors of sporadic colorectal cancers (CRCs) with microsatellite instability (MSI), and to be tightly associated with BRAF mutation and the CpG island methylator phenotype (CIMP). Consequently, colonoscopic identification of SSAs has important implications for preventing CRCs, but accurate endoscopic diagnosis is often difficult. Our aim was to clarify which endoscopic findings are specific to SSAs. METHODS: The morphological, histological and molecular features of 261 specimens from 226 colorectal tumors were analyzed. Surface microstructures were analyzed using magnifying endoscopy. Mutation in BRAF and KRAS was examined by pyrosequencing. Methylation of p16, IGFBP7, MLH1 and MINT1, -2, -12 and -31 was analyzed using bisulfite pyrosequencing. RESULTS: Through retrospective analysis of a training set (n=145), we identified a novel surface microstructure, the Type II open-shape pit pattern (Type II-O), which was specific to SSAs with BRAF mutation and CIMP. Subsequent prospective analysis of an independent validation set (n=116) confirmed that the Type II-O pattern is highly predictive of SSAs (sensitivity, 65.5%; specificity, 97.3%). BRAF mutation and CIMP occurred with significant frequency in Type II-O-positive serrated lesions. Progression of SSAs to more advanced lesions was associated with further accumulation of aberrant DNA methylation and additional morphological changes, including the Type III, IV and V pit patterns. CONCLUSIONS: Our results suggest the Type II-O pit pattern is a useful hallmark of the premalignant stage of CRCs with MSI and CIMP, which could serve to improve the efficacy of colonoscopic surveillance.
Assuntos
Adenoma/patologia , Neoplasias Colorretais/patologia , Lesões Pré-Cancerosas/patologia , Adenoma/genética , Idoso , Análise de Variância , Distribuição de Qui-Quadrado , Colonoscopia , Neoplasias Colorretais/genética , Ilhas de CpG/genética , Metilação de DNA , Análise Mutacional de DNA , Progressão da Doença , Feminino , Humanos , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Fenótipo , Lesões Pré-Cancerosas/genética , Valor Preditivo dos Testes , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras) , Curva ROC , Estudos Retrospectivos , Sensibilidade e Especificidade , Proteínas ras/genéticaRESUMO
Although minimal invasive treatment is widely accepted in the early stages of gastric cancer (GCa), we still do not have any appropriate risk markers to detect residual neoplasia and the potential for recurrence. We previously reported that aberrant DNA methylation is an early and frequent process in gastric carcinogenesis and could be useful for the detection of gastric neoplasia. Our goal is to find and identify some candidate genes, using genome-wide DNA methylation analysis, as a treatment marker for early gastric cancer (EGC). We performed methylated CpG island amplification microarray analysis using 12 gastric washes (six each of pre- and post-endoscopic treatment in each of the same patients). We finally focused on Sox17 gene. We examined the DNA methylation status of Sox17 in a validation set consisting of 128 wash samples (pre, 64; post, 64) at EGC. We next carried out functional studies to identify Sox17. Sox17 showed significant differential methylation between pre- and post-treatments in EGC patients (Sox17, p < 0.0001). Moreover, treating GCa cells that lacked Sox17 expression with a methyltransferase inhibitor, 5-aza-2'-deoxycytidine, restored the gene's expression. Additionally, the introduction of exogenous Sox17 into silenced cells suppressed colony formation. Gastric wash-based DNA methylation analysis could be useful for early detection of recurrence following endoscopic resection in EGC patients. Our data suggest that the silencing of Sox17 occurs frequently in EGC and may play a key role in the development and progression of the disease.
Assuntos
Biomarcadores Tumorais/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição SOXF/genética , Neoplasias Gástricas/genética , Idoso , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linhagem Celular Tumoral , Ilhas de CpG , Decitabina , Progressão da Doença , Detecção Precoce de Câncer/métodos , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Pessoa de Meia-Idade , RiscoRESUMO
Promoter CpG island hypermethylation of tumor suppressor genes is a common hallmark of all human cancers. Many researchers have been looking for potential epigenetic therapeutic targets in cancer using gene expression profiling with DNA microarray approaches. Our recent genome-wide platform of CpG island hypermethylation and gene expression in colorectal cancer (CRC) cell lines revealed that FBN2 and TCERG1L gene silencing is associated with DNA hypermethylation of a CpG island in the promoter region. In this study, promoter DNA hypermethylation of FBN2 and TCERG1L in CRC occurs as an early and cancer-specific event in colorectal cancer. Both genes showed high frequency of methylation in colon cancer cell lines (>80% for both of genes), adenomas (77% for FBN2, 90% for TCERG1L, n = 39), and carcinomas (86% for FBN2, 99% for TCERG1L, n = 124). Bisulfite sequencing confirmed cancer-specific methylation of FBN2 and TCERG1L of promoters in colon cancer cell line and cancers but not in normal colon. Methylation of FBN2 and TCERG1L is accompanied by downregulation in cell lines and in primary tumors as described in the Oncomine™ website. Together, our results suggest that gene silencing of FBN2 and TCERG1L is associated with promoter DNA hypermethylation in CRC tumors and may be excellent biomarkers for the early detection of CRC.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias do Colo/genética , Metilação de DNA , Detecção Precoce de Câncer/métodos , Idoso , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Ilhas de CpG , Primers do DNA/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras GenéticasRESUMO
Aberrant DNA methylation has been implicated in the development of hepatocellular carcinoma (HCC). Our aim was to clarify its molecular mechanism and to identify useful biomarkers by screening for DNA methylation in HCC. Methylated CpG island amplification coupled with CpG island microarray (MCAM) analysis was carried out to screen for methylated genes in primary HCC specimens [hepatitis B virus (HBV)-positive, n = 4; hepatitis C virus (HCV)-positive, n = 5; HBV/HCV-negative, n = 7]. Bisulfite pyrosequencing was used to analyze the methylation of selected genes and long interspersed nuclear element (LINE)-1 in HCC tissue (n = 57) and noncancerous liver tissue (n = 50) from HCC patients and in HCC cell lines (n = 10). MCAM analysis identified 332, 342, and 259 genes that were methylated in HBV-positive, HCV-positive, and HBV/HCV-negative HCC tissues, respectively. Among these genes, methylation of KLHL35, PAX5, PENK, and SPDYA was significantly higher in HCC tissue than in noncancerous liver tissue, irrespective of the hepatitis virus status. LINE-1 hypomethylation was also prevalent in HCC and correlated positively with KLHL35 and SPDYA methylation. Receiver operating characteristic curve analysis revealed that methylation of the four genes and LINE-1 strongly discriminated between HCC tissue and noncancerous liver tissue. Our data suggest that aberrant hyper- and hypomethylation may contribute to a common pathogenesis mechanism in HCC. Hypermethylation of KLHL35, PAX, PENK, and SDPYA and hypomethylation of LINE-1 could be useful biomarkers for the detection of HCC.
Assuntos
Carcinoma Hepatocelular/genética , Metilação de DNA , Neoplasias Hepáticas/genética , Adulto , Idoso , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Ilhas de CpG , Epigênese Genética , Feminino , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Elementos Nucleotídeos Longos e Dispersos , Masculino , Pessoa de Meia-Idade , Fator de Transcrição PAX5/genéticaRESUMO
Gimeracil (5-chloro-2, 4-dihydroxypyridine) is an inhibitor of dihydropyrimidine dehydrogenase (DPYD), which degrades pyrimidine including 5-fluorouracil in the blood. Gimeracil was originally added to an oral fluoropyrimidine derivative S-1 to yield prolonged 5-fluorouracil concentrations in serum and tumor tissues. We have already reported that gimeracil had radiosensitizing effects by partially inhibiting homologous recombination (HR) in the repair of DNA double strand breaks. We investigated the mechanisms of gimeracil radiosensitization. Comet assay and radiation-induced focus formation of various kinds of proteins involved in HR was carried out. siRNA for DPYD were transfected to HeLa cells to investigate the target protein for radiosensitization with gimeracil. SCneo assay was carried out to examine whether DPYD depletion by siRNA inhibited HR repair of DNA double strand breaks. Tail moments in neutral comet assay increased in gimeracil-treated cells. Gimeracil restrained the formation of foci of Rad51 and replication protein A (RPA), whereas it increased the number of foci of Nbs1, Mre11, Rad50, and FancD2. When HeLa cells were transfected with the DPYD siRNA before irradiation, the cells became more radiosensitive. The degree of radiosensitization by transfection of DPYD siRNA was similar to that of gimeracil. Gimeracil did not sensitize DPYD-depleted cells. Depletion of DPYD by siRNA significantly reduced the frequency of neopositive clones in SCneo assay. Gimeracil partially inhibits the early step in HR. It was found that DPYD is the target protein for radiosensitization by gimeracil. The inhibitors of DPYD, such as gimeracil, could enhance the efficacy of radiotherapy through partial suppression of HR-mediated DNA repair.
Assuntos
Reparo do DNA , Di-Hidrouracila Desidrogenase (NADP)/antagonistas & inibidores , Piridinas/farmacologia , Radiossensibilizantes/farmacologia , Recombinação Genética , Linhagem Celular Tumoral , Inibidores Enzimáticos , Células HeLa , Humanos , RNA Interferente Pequeno/farmacologia , TransfecçãoRESUMO
OBJECTIVES: Endoscopic examination shows that serrated neoplasias (SNs), such as serrated adenomas and sessile serrated adenomas, exhibit different mucosal crypt patterns. However, it remains unclear whether advanced serrated polyps with different mucosal crypt patterns have different clinicopathological or molecular features. METHODS: We classified the mucosal crypt patterns of 86 SNs into three types (hyperplastic, adenomatous, and mixed pattern) and evaluated their clinicopathological and molecular features. RESULTS: We found significant differences in the proliferative activity status between SNs with mixed/adenomatous patterns and those with the hyperplastic patterns. SNs with the hyperplastic pattern were frequently located in the proximal colon and had a macroscopically superficial appearance, whereas SNs with the adenomatous pattern were often located in the distal colon and had a protruding appearance. Furthermore, a significant difference was observed in the frequency of the CpG island methylator phenotype (CIMP), involving the methylation of two or more CIMP-related genes (MINT1, MINT2, MINT31, p16, and MLH1), between SNs with the hyperplastic pattern and those with the mixed/adenomatous patterns (18/32 (56%) vs. 8/28 (29%) or 7/26 (27%); P=0.0309 or P=0.0249, respectively). Moreover, the prevalence of KRAS mutations was significantly higher in SNs with the adenomatous pattern than in those with the hyperplastic pattern (7/26 (27%) vs. 1/32 (3%); P=0.0173). In comparison with other patterns, the mixed pattern was detected more frequently in mixed serrated polyps (MSPs), which contain separate histological components. Some MSPs exhibited concordant molecular alterations among the different histological components. CONCLUSIONS: The clinicopathological and molecular features of SNs correlated strongly with their mucosal crypt patterns, which were observed using chromoendoscopy.
Assuntos
Pólipos Adenomatosos/patologia , Pólipos do Colo/patologia , Neoplasias Colorretais/patologia , Hiperplasia/patologia , Mucosa Intestinal/patologia , Lesões Pré-Cancerosas/patologia , Pólipos Adenomatosos/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Proliferação de Células , Distribuição de Qui-Quadrado , Pólipos do Colo/genética , Neoplasias Colorretais/genética , Ilhas de CpG/genética , Feminino , Humanos , Hiperplasia/genética , Mucosa Intestinal/metabolismo , Antígeno Ki-67/metabolismo , Masculino , Metilação , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Lesões Pré-Cancerosas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras) , Estatísticas não Paramétricas , Adulto Jovem , Proteínas ras/genéticaRESUMO
Cancer cells exhibit two opposing methylation abnormalities: genome-wide hypomethylation and gene promoter hypermethylation. Downregulation of E-cadherin (CDH1) plays a key role in the development of diffuse-type gastric cancer, and DNA methylation is a major cause of the gene's silencing. Hereditary diffuse gastric cancer is caused by germline mutation of CDH1 gene, and DNA methylation frequently serves as the second hit completely inactivating the gene. In sporadic diffuse-type gastric cancer, methylation of CDH1 is more prevalent than mutation of the gene. Epstein-Barr virus (EBV)-associated gastric carcinoma (EBV-associated GC) is characterized by concurrent methylation of multiple genes, and diffuse-type gastric cancer is frequently seen among EBV-associated GCs. Patients with pangastritis or enlarged-fold gastritis, which are both caused by Helicobacter pylori infection, reportedly have an increased risk for diffuse-type gastric cancer. Notably, the gastric mucosa of enlarged-fold gastritis patients exhibits CDH1 hypermethylation and genome-wide hypomethylation. These data suggest that aberrant DNA methylation is an essential promoter of carcinogenesis in individuals at high risk for diffuse-type gastric cancer.
Assuntos
Metilação de DNA , DNA de Neoplasias/genética , Neoplasias Gástricas/genética , Antígenos CD , Caderinas/genética , Inativação Gênica , HumanosRESUMO
BACKGROUND: A number of noninvasive tests have been developed to establish the presence of Helicobacter pylori infection. However, thus far these tests have only been capable of detecting its presence. An increasing number of antibiotic-resistant H. pylori infections have been reported and they are known to be correlated with 23S rRNA single nucleotide polymorphisms (SNPs). We hypothesized that genomic analysis of H. pylori recovered from gastric washes could not only be less invasive, but also useful as a screening test and for assessing the outcome of eradication therapy. METHODS: Biopsy specimens and gastric washes were collected from 100 patients during endoscopic examination. Then we analyzed 23S rRNA, ureA, and cagA genes using PCR and high-throughput pyrosequencing analysis. RESULTS: Forty-five percent (44/97) of patients tested positive for ureA and 42.3% (41/97) tested positive by a rapid urease test. One hundred percent (35/35) of patients who tested positive by both methods were observed to have the cagA gene. Among these 35 patients, 23S rRNA SNPs were present in 34.3% (12/35). CONCLUSIONS: Gastric wash-based PCR and a pyrosequencing assay were used to rapidly detect and estimate the number of 23S rRNA SNPs in clinical isolates of H. pylori. Not only is this a less invasive technique, but it can also diagnose drug resistance.
Assuntos
Farmacorresistência Bacteriana/genética , Mucosa Gástrica/patologia , Infecções por Helicobacter/diagnóstico , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori/genética , RNA Ribossômico 23S/genética , 2-Piridinilmetilsulfinilbenzimidazóis/uso terapêutico , Adulto , Idoso , Amoxicilina/uso terapêutico , Antibacterianos/uso terapêutico , Antiulcerosos/uso terapêutico , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Biópsia , Claritromicina/uso terapêutico , Feminino , Lavagem Gástrica , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Rabeprazol , Análise de Sequência de RNA , Estatísticas não Paramétricas , Urease/genética , Urease/metabolismoRESUMO
Altered expression of microRNA (miRNA) is strongly implicated in cancer, and recent studies have shown that the silencing of some miRNAs is associated with CpG island hypermethylation. To identify epigenetically silenced miRNAs in gastric cancer (GC), we screened for miRNAs induced by treatment with 5-aza-2'-deoxycytidine and 4-phenylbutyrate. We found that miR-34b and miR-34c are epigenetically silenced in GC and that their downregulation is associated with hypermethylation of the neighboring CpG island. Methylation of the miR-34b/c CpG island was frequently observed in GC cell lines (13/13, 100%) but not in normal gastric mucosa from Helicobacter pylori-negative healthy individuals. Transfection of a precursor of miR-34b and miR-34c into GC cells induced growth suppression and dramatically changed the gene expression profile. Methylation of miR-34b/c was found in a majority of primary GC specimens (83/118, 70%). Notably, analysis of non-cancerous gastric mucosae from GC patients (n = 109) and healthy individuals (n = 85) revealed that methylation levels are higher in gastric mucosae from patients with multiple GC than in mucosae from patients with single GC (27.3 versus 20.8%; P < 0.001) or mucosae from H. pylori-positive healthy individuals (27.3 versus 20.7%; P < 0.001). These results suggest that miR-34b and miR-34c are novel tumor suppressors frequently silenced by DNA methylation in GC, that methylation of miR-34b/c is involved in an epigenetic field defect and that the methylation might be a predictive marker of GC risk.
Assuntos
Metilação de DNA , Inativação Gênica , MicroRNAs/antagonistas & inibidores , Neoplasias Gástricas/genética , Linhagem Celular Tumoral , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/análise , MicroRNAs/fisiologia , Análise de Sequência com Séries de OligonucleotídeosRESUMO
A subset of colorectal cancers (CRCs) show simultaneous methylation of multiple genes; these tumors have the CpG island methylator phenotype (CIMP). CRCs with CIMP show a specific pattern of genetic alterations, including a high frequency of BRAF mutations and a low frequency of p53 mutations. We therefore hypothesized that genes inactivated by DNA methylation are involved in the BRAF- and p53-signaling pathways. Among those, we examined the epigenetic inactivation of insulin-like growth factor-binding protein 7 (IGFBP7) expression in CRCs. We found that in CRC cell lines, the silencing of IGFBP7 expression was correlated with high levels of DNA methylation and low levels of histone H3K4 methylation. Luciferase and chromatin immunoprecipitation assays in unmethylated cells revealed that p53 induces expression of IGFBP7 upon binding to a p53 response element within intron 1 of the gene. Treating methylated CRC cell lines with 5-aza-2'-deoxycytidine restored p53-induced IGFBP7 expression. Levels of IGFBP7 methylation were also significantly higher in primary CRC specimens than in normal colonic tissue (P < 0.001). Methylation of IGFBP7 was correlated with BRAF mutations, an absence of p53 mutations and the presence of CIMP. Thus, epigenetic inactivation of IGFBP7 appears to play a key role in tumorigenesis of CRCs with CIMP by enabling escape from p53-induced senescence.
Assuntos
Adenocarcinoma/genética , Neoplasias Colorretais/genética , Ilhas de CpG , Metilação de DNA , Inativação Gênica , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/fisiologia , Proteínas de Neoplasias/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Adenocarcinoma/patologia , Adenoma/genética , Adenoma/patologia , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Senescência Celular , Neoplasias Colorretais/patologia , Sequência Consenso , Metilação de DNA/efeitos dos fármacos , DNA de Neoplasias/química , DNA de Neoplasias/genética , Decitabina , Genes p53 , Histonas/genética , Humanos , Fenótipo , Proteínas Proto-Oncogênicas B-raf/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Ensaio Tumoral de Célula-TroncoRESUMO
The Ras/B-Raf/C-Raf/MEK/ERK signaling cascade is critical for the control of many fundamental cellular processes, including proliferation, survival, and differentiation. This study demonstrated that small interfering RNA-dependent knockdown of diacylglycerol kinase eta (DGKeta) impaired the Ras/B-Raf/C-Raf/MEK/ERK pathway activated by epidermal growth factor (EGF) in HeLa cells. Conversely, the overexpression of DGKeta1 could activate the Ras/B-Raf/C-Raf/MEK/ERK pathway in a DGK activity-independent manner, suggesting that DGKeta serves as a scaffold/adaptor protein. By determining the activity of all the components of the pathway in DGKeta-silenced HeLa cells, this study revealed that DGKeta activated C-Raf but not B-Raf. Moreover, this study demonstrated that DGKeta enhanced EGF-induced heterodimerization of C-Raf with B-Raf, which transmits the signal to C-Raf. DGKeta physically interacted with B-Raf and C-Raf, regulating EGF-induced recruitment of B-Raf and C-Raf from the cytosol to membranes. The DGKeta-dependent activation of C-Raf occurred downstream or independently of the already known C-Raf modifications, such as dephosphorylation at Ser-259, phosphorylation at Ser-338, and interaction with 14-3-3 protein. Taken together, the results obtained strongly support that DGKeta acts as a novel critical regulatory component of the Ras/B-Raf/C-Raf/MEK/ERK signaling cascade via a previously unidentified mechanism.